Journal of Chromatography A, 919 (2001) 13–19

www.elsevier.com / locate / chroma

Polymer characterization by size-exclusion chromatography with

multiple detection q
M. Teresa R. Laguna, Ricardo Medrano, Miguel P. Plana, M. Pilar Tarazona*
´
Departamento de Quımica ´
Fısica , Universidad de Alcala´ , 28871 Alcala´ de Henares, Spain

Received 20 November 2000; received in revised form 22 March 2001; accepted 27 March 2001

Abstract

Size-exclusion chromatography with coupled multiangle light scattering and differential refractometry detectors has been
used to obtain molecular mass and radius of gyration distributions of polydisperse polymer samples. From these data the
scaling law between dimensions and the absolute molecular mass is obtained with just one sample of each polymer. Three
different kinds of polymers are presented: polystyrene which serves as reference polymer, polyphosphazenes which behave
abnormally in solution and poly(ethylene oxide) which is soluble in water. Since the relationship between dimensions and
molecular mass depends on the extent of interactions between chain segments and solvent molecules, the scaling law
provides information about the solution properties of the polymer.  2001 Elsevier Science B.V. All rights reserved.

Keywords: Multiangle light scattering detection; Detection, LC; Polymers; Polystyrene; Polyphosphazenes; Poly(ethylene
oxide)

1. Introduction molar size distribution. However, the size of the

Size-exclusion chromatography (SEC) is the most
common way to obtain information about the molec-
ular mass distribution of polymers. In this technique,
it is often supposed that there is an absence of
interaction between the macromolecules and the gel
contained in the columns, and consequently, mole-
cules are separated according to their size, yielding a
q
Presented at the 29th Scientific Meeting of the Spanish Group
of Chromatography and Related Techniques, Alcala´ de Henares
(Madrid), 12–14 July, 2000.
*Corresponding author. Tel.: 134-918-854-664; fax: 134-918-
854-763.
E-mail address: mpilar.tarazona@uah.es (M.P. Tarazona).
polymer depends not only on its molecular mass but
also on its physical architecture distribution, thus a
calibration with narrow polydispersity index stan-
dards of the same polymer is required in order to
obtain true molecular mass distributions. Unfortuna-
tely, suitable monodisperse standards are not always
available so that other approaches like universal
calibration or numerical procedures using broad
polydisperse samples, must be used [1,2].
The use of a molecular mass sensitive detection
method, such as multiangle light scattering (MALS)
detection, coupled to a customary concentration
detection method, such as the conventional refractive
index (RI) detection, allows absolute molecular
masses determination for each slice across a sample
peak. Thus, it offers the possibility of analyzing a

0021-9673 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0021-9673( 01 )00802-0
14 M.T.R. Laguna et al. / J. Chromatogr. A 919 (2001) 13–19
polymer sample in terms of the absolute distribution
of molecular mass [3–8].
Furthermore, the angular dependence of the scat-
tered intensity may be determined at each elution
volume. Thus, the light scattering technique also
provides a direct measurement of the polymer di-
mensions, the mean square radius of gyration, ks 2 l.
The on-line MALS detection method presents the
appealing possibility of being able to obtain the
relationship between molecular mass and dimen-
sions, i.e. the scaling law, with just one sample for
broad molecular mass distribution polymers. Since
the polymer dimensions depend on the polymer–
solvent interactions, the scaling law provides in-
formation about the solution properties of the poly-
mer [9–12]. This technique can be carried out with
minimal sample preparation, thus avoiding tedious
and time consuming polymer fractionation, and can
greatly speed up the elucidation of polymer solution
properties.
This work presents the results obtained for differ-
ent polymers using SEC with a MALS photometer
and a differential refractometer (RI) as detectors.
Two kinds of polymers, lipophilic polymers: poly-
styrene (PS), poly[(diphenoxy)phosphazene] (PDPP)
and poly[di(b-naftoxy)phosphazene) (PDNP) and a
hydrophilic polymer, poly(ethylene oxide) (PEO)
were studied by means of organic and aqueous SEC,
respectively (Fig. 1). PS was chosen since it is a
well-behaving polymer commonly used as a refer-
ence polymer [13]. On the contrary, the solution
behavior of polyphosphazenes is anomalous, they do
not always follow the universal calibration approach
in SEC and its fractionation is very difficult
[11,14,15]. PEO is a water-soluble polymer whose
conformational properties in water depend on the
presence of salts [16,17].
Fig. 1. Polymer structures.
2. Experimental
SEC measurements were carried out using Waters
Associate equipment with a model 510 pump, a U6K
injector, a differential RI detector model 410. A
Dawn-DSP-F MALS photometer operating at 632.8
nm from Wyatt Technology was the molecular mass
and size detector used. The measurements were
carried out at 258C. Calibration of the Dawn equip-
ment was done using toluene and the normalization
of the detectors was performed with standard mono-
disperse PS and PEO of low molecular mass that did
not show angular dependence on the light scattering
signal. The above standard polymers were also used
to determine the interdetector volume [5]. For or-
ganic SEC, two columns PLgel mixed B (Polymer
Labs.) in series were used, whereas two Ul-
trahydrogel Linear 6–13 mm were used for aqueous
SEC. The eluent was tetrahydrofuran (THF) freshly
distilled from sodium and benzophenone for PS;
THF with a 0.1% tetra-n-butylammonium bromide
for PDPP and PDNP; water and a 0.1 M NaNO 3
aqueous solution for PEO. The eluents were filtered
through a 0.2-mm filter and degassed. The flow-rates
were 1.0 and 0.5 ml / min for organic and aqueous
SEC, respectively.
PDPP and PEO were commercial samples (Al-
drich). PS and PDNP were synthesized by bulk
polymerization of styrene and hexachlorocyclot-
riphosphazene, respectively, as explained elsewhere
[15,18].
The software used, ASTRA 4.73 from Wyatt Tech-
nology, allowed on-line data of molecular mass and
radius of gyration collection as well as calculation of
the distributions and averages.
The differential refractive index increments for the
polymers in the solvents were taken from literature
[13] or measured with a Brice-Phoenix differential
refractometer or an Optilab-DSP interferometric
refractometer.
3. Theory
The basic equation when using a light scattering
detector [5,19] is:
Kc 1
]] 5 ] 1 1 ]]
DRu Mw S
16p 2 2
3l 2 ks l sin
2 u
SD
] 1 ....
2 D (1)
 M.T.R. Laguna et al. / J. Chromatogr. A 919 (2001) 13–19 15

where DRu is the excess Rayleigh ratio, related to the

scattered light intensity of the polymer at angle of
observation u, c is the concentration of the polymer
solution, l the light wavelength, ks 2 l the mean
square radius of gyration, Mw the weight-average
molecular mass, and K is the optical constant. K is
related to the wavelength in vacuum, l0 , the solvent
refraction index, n, Avogadro’s number, NA , and
refraction index increment, dn / dc by:

K 5 ]] S D
4p 2 n 2 dn
]
l 40 NA dc
2
(2)

The MALS detector measures simultaneously the

excess Rayleigh ratio for each slice i at different u
angles and the slices are assumed to be monodisperse
both in composition and molecular mass [5,6]. Thus,
a plot of Kc /DRu versus sin 2 (u / 2), affords to
calculate the molecular mass Mi from the intercept
and the mean square radius of gyration, ks 2 l i , from
the slope for each slice of the chromatogram.
The signal of the RI detector is proportional to the
polymer concentration and to the refractive index
increment [8]. Thus, for each slice: Fig. 2. Logarithm of molecular mass and root mean squared
radius of gyration versus elution volume for polystyrene and
S Ddn
S RI 5 k RI c ]
dc
(3) polyphosphazenes. The corresponding RI signals are also shown.

where k RI is the absolute response factor of the

detector that does not depend on the kind of polymer
and can be assumed to be constant and c is the
polymer concentration in the slice. When using the
RI detector the concentration c will be the true
concentration if the dn / dc of the slice coincides with
the dn / dc of the whole sample.
4. Results and discussion
4.1. Organic size-exclusion chromatography
Fig. 2 shows the molecular mass and the root
mean squared radius of gyration for PS, PDPP and
PDNP, versus the elution volume, ve , i.e. the abso-
lute calibration curves. The corresponding signals of
the RI detector for the chromatograms are superim-
posed. At the edges of the peaks, the signal-to-noise
ratio is low and there is a larger uncertainty, espe-
cially for high elution volumes. PS shows linear
calibration curves of M and ks 2 l 1 / 2 over the entire
range of molecular mass, whereas upward curvatures
are observed for polyphosphazenes at high elution
volumes. This curvature, has been found in poly-
phosphazenes [14,20] and other types of polymers
[21,22] and indicates the existence of a small frac-
tion of molecules, which are retarded in the columns.
This fact may lead to errors if universal calibration
[23] is used to obtain molecular masses, whereas
using a light scattering detector it is easily visual-
ized. In spite of being similar polymers studied under
the same conditions, PDPP and PDNP have very
different calibration curves.
At the very low concentrations used in SEC, the
calculation of the values for the z average root mean
square radius of gyration ks 2 l 1 / 2 is independent [24]
of both dn / dc and M and therefore, it is an excellent
parameter to study the dimensions of the polymer.
However, the size of the polymer must be larger than
| l / 20 in order to observe the angular dependence of
the scattered light intensity [19]. Thus the accuracy
of the radius of gyration, obtained in the region of
16 M.T.R. Laguna et al. / J. Chromatogr. A 919 (2001) 13–19
Fig. 3. Molecular mass distributions for polystyrene and poly-
phosphazenes.
low molecular masses, begins to deteriorate rapidly
for PDNP, the sample with lower-molecular-mass
chains (Fig. 2).
Fig. 3 presents the absolute molecular mass dis-
tributions (MMDs) of the polymers calculated from
the combined measurements of molecular mass
(MALS detector) and concentration (RI detector).
Different molecular mass averages and the polydis-
persity index (Mw /Mn , where Mn is the number-
average molecular mass), calculated from the dis-
tributions, are presented in Table 1. The three
samples are polydisperse covering a broad range of
molecular masses. The MMD of PDNP is more
displaced to low molecular masses, as explained
above.
Since the three samples are polydisperse and cover
an ample range of molecular masses, the relationship
between the polymer dimensions and the molecular
mass can be obtained for each sample. This relation-
ship is usually described as a scaling law of the form
[9]:
Table 1
Molecular mass averages and polydispersities for the polymer samples
10 25 Mn
PS 9.860.1
PDPP 9.4160.01
PDNP 3.6460.01
PEO (water) 9.662.0
PEO (0.1 M NaNO 3 ) 1063
Fig. 4. Log–log plot of root mean squared radius of gyration
versus molecular mass, i.e. scaling laws, for the polymers in the
whole range of molecular masses. The linear fits corresponding to
the scaling parameters of Table 2 are also shown.
ks 2 l 1 / 2 5 QM q (4)
and yields information about the solution properties
of the polymer. The power law exponent q is related
to the shape of the chain and yields information
about the polymer–solvent interactions and macro-
molecular conformation of the polymer. Values of
0.5 # q # 0.6 are predicted for random-coil chains,
from polymers in theta conditions (0.5) to polymers
in very good solvents (0.6). Rigid rod polymers
present values of q as high as 1 whereas spherical
particles have a q value equal to 1 / 3.
Fig. 4 shows the log–log plot of the scaling law
(Eq. (4)) obtained for the polymer samples and the
values of the q and Q parameters are shown in Table
2. For PS the plot is linear and the value of q¯0.6
indicates that, as expected, PS chains are in random
coil conformation and expanded due to the excluded
volume effect since THF is a very good solvent for
this polymer. For the polyphosphazenes, PDPP and
10 26 Mw 10 26 Mz Mw /Mn
1.6760.03 4.560.7 1.7060.04
1.8960.01 3.9460.01 2.0160.01
0.97660.001 2.8960.01 2.6860.01
1.860.3 3.562.0 1.960.5
1.760.4 5.066.0 1.660.6
 M.T.R. Laguna et al. / J. Chromatogr. A 919 (2001) 13–19 17

Table 2 Fig. 5 presents plots similar to Fig. 2, i.e. cali-

Scaling law parameters
10 3 Q q temperature and rate of elution, but two different
PS 12.460.3 0.5960.01
PDPP 8163 0.4360.01
PDNP 8561 0.406 0.01
PEO (water) 1.1560.09 0.7560.01
PEO (0.1 M NaNO 3 ) 177614 0.4260.01
PDNP, the plots show a pronounced curvature at low
molecular masses as a consequence of the curvatures
showed in the calibration curves (Fig. 2) explained
above [14]. The values of q obtained for both
polyphosphazenes are lower than 0.5 thus, THF is a
bad solvent, the polymers are under u conditions and
adopt more compact conformations. The results
obtained for PDNP presented in this work are in
good agreement with our previous results obtained
through a much more laborious procedure [15]. The
method consisted firstly in fractionation of the
polymer, secondly in SEC, light scattering and
viscosity measurements of the samples and thirdly
since the fractionation yielded broad samples, in the
use of numerical methods of adjustment [15]. The
coupled SEC–MALS technique used in this work, is
not only much simpler but also explains, from the
plots of Figs. 2 and 4, why the fractionation was not
effective.
bration curves for the PEO using the same columns,
eluents, water and a 0.1 M aqueous solution of
sodium nitrate. The shapes of the SEC chromato-
grams and the slope of the molecular mass cali-
bration curves in the two eluents are similar. How-
ever, the elution volumes increase markedly if salt is
present, indicating a decrease of the hydrodynamic
volume of the polymer when salt is added. In the
lower part of Fig. 5, the calibration curves of the
radius of gyration show a different slope indicating a
change in the conformation of the polymer which
can be the reason for the decrease of the hydro-
dynamic volume. This change will be more easily
observed in the scaling law that will be explained
below.
Fig. 6 shows the absolute molecular mass dis-
tributions obtained with the two solvents and the
averaged molecular masses are presented in the last

4.2. Aqueous size-exclusion chromatography

Aqueous SEC permits the determination of the

molecular mass distribution of water-soluble poly-
mers. The principal separation mechanism is similar
to conventional SEC; thus, smaller polymers have
more pore volume available and elute later than
larger polymers. The solvents used as eluents are
water, polar solvents or ionic solutions and since the
polar interactions between polymers, eluent and
support can be significant, additional separation
mechanisms, besides the steric exclusion one, can
influence the separation process. A lot of effort is
being made towards the understanding and im-
plementation of the technique [4,25] and, since the
ionic strength of the solvent can affect the polymer
conformation, the use of the light scattering detector Fig. 5. Logarithm of molecular mass and root mean squared
can provide information about this conformational radius of gyration versus elution volume for PEO in the two
change. eluents used. The corresponding RI signals are also shown.
18 M.T.R. Laguna et al. / J. Chromatogr. A 919 (2001) 13–19
Fig. 6. Molecular mass distributions for PEO.
two rows of Table 1. They are similar within
experimental error. Comparing the Figs. 2 and 5, the
uncertainty in the calibration curves is larger for
aqueous SEC due to the fact that the signal-to-noise
ratio is lower in aqueous SEC than in organic SEC.
The scaling laws for PEO in the two solvents are
presented in Fig. 7 and the scaling parameters are
shown in Table 2. Although the uncertainty in the
measurements is large, it is clear that the scaling law
and thus the polymer conformation strongly depends
on the addition of NaNO 3 . The value of the q
Fig. 7. Log–log plot of root mean squared radius of gyration
versus molecular mass, i.e. scaling laws, for PEO in the two
eluents. The linear fits corresponding to the scaling parameters of
Table 2 are also shown.
parameter obtained for PEO in water, 0.75, is
considerably higher than the value corresponding to
a random coil polymer in a good solvent, 0.6. Thus
the chain is further extended than a random coil
polymer and there is a tendency of the chains to
adopt rod type conformations. However, if NaNO 3 is
added, the polymer chain contracts up to values well
below u conditions, and close to a spherical shape.
These results agree with viscosity measurements and
theoretical calculations. It has been reported that the
addition of sodium or potassium salts to water
reduces the viscosity of PEO of high molecular mass
[26,27] and also lowers the u temperature [13].
Tasaki performed molecular dynamic simulations of
PEO chains in water; the chain showed a preference
for helical conformations stabilized by hydrogen
bonds with water molecules [28]. Moreover the
addition of K 1 disrupted the hydrogen bonds and
considerably distorted the helical conformation of the
chain [16].
5. Conclusions
The use of a SEC–MALS–RI chromatographic
system gives information about conformational prop-
erties of polymers in solution using just one broad
polydisperse sample of the polymer. The technique
can be applied to both lipophilic and hydrophilic
polymers using organic and aqueous SEC, respec-
tively. Moreover, the method is fast and avoids
tedious and time consuming polymer fractionation.
The results obtained for a standard polymer like
PS are similar to those expected whereas the anomal-
ous behavior of polyphosphazenes PDPP is ob-
served. In aqueous SEC, although the accuracy of the
measurements is lower, the differences in the PEO
conformations when a salt is added can be de-
termined through the two different scaling law
exponents obtained.
Acknowledgements
Financial support from DGICYT through project
PB97-0778 is gratefully acknowledged.
 M.T.R. Laguna et al. / J. Chromatogr. A 919 (2001) 13–19
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